Suppressor mutations in
            <i>Escherichia coli</i>
            methionyl-tRNA formyltransferase: Role of a 16-amino acid insertion  module in initiator tRNA recognition

Ramesh, V.; Gite, Sadanand; Li, Yan; RajBhandary, Uttam L.

doi:10.1073/pnas.94.25.13524

Cited by 29 publications

(37 citation statements)

References 44 publications

(50 reference statements)

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“…Ribosomal protein RpsB and Fmt, which catalyzes the reaction of formylation of initiator methionyltRNA, were up-regulated in respect to the exponential and stationary phases. Fmt was previously reported to be important for the initiation of protein synthesis (11,27). Induction of elongation factors FusA and Tuf was observed in relation to the stationary phase.…”

Section: Discussionmentioning

confidence: 81%

Differential Expression of Proteins and Genes in the Lag Phase of Lactococcus lactis subsp. lactis Grown in Synthetic Medium and Reconstituted Skim Milk

Larsen

Boye

Siegumfeldt

et al. 2006

Appl Environ Microbiol

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We investigated protein and gene expression in the lag phase of Lactococcus lactis subsp. lactis CNRZ 157 and compared it to the exponential and stationary phases. By means of two-dimensional polyacrylamide gel electrophoresis, 28 highly expressed lag-phase proteins, implicated in nucleotide metabolism, glycolysis, stress response, translation, transcription, cell division, amino acid metabolism, and coenzyme synthesis, were identified. Among the identified proteins, >2-fold induction and down-regulation in the lag phase were determined for 12 proteins in respect to the exponential phase and for 18 proteins in respect to the stationary phase. Transcriptional changes of the lag-phase proteins in L. lactis were studied by oligonucleotide microarrays. Good correlation between protein and gene expression studies was demonstrated for several differentially expressed proteins, including nucleotide biosynthetic enzymes, adenylosuccinate synthase (PurA), IMP dehydrogenase (GuaB), and aspartate carbamoyl transferase (PyrB); heat-shock protein DnaK; serine hydroxymethyl transferase (GlyA); carbon catabolite control protein (CcpA); elongation factor G (FusA); and cell division protein (FtsZ).Intracellular events occurring in bacteria just after introduction to a new environment provide the first evidence about physiological changes in the cells. When lactic acid bacteria are used as starter cultures, these intracellular changes may ensure a rapid prediction of the overall physiological status of the culture, which in turn defines the activity of the culture. Upon inoculation, the culture is exposed to massive environmental changes including nutrient availability, temperature, and pH. To adapt to new environments, bacteria have developed various metabolic responses. Recent research revealed a number of responses in Lactococcus lactis caused by exhaustion of essential nutrients such as purines (1), carbohydrates (7,18,36), and branched amino acids (10, 29), as well as responses to the temperature shift (6, 42), low pH (4,23,26,31), and a combination of these factors (13,25). However, only a few papers describe the intracellular changes occurring early in the cultivation upon transfer of the culture to a new rich medium. For example, proteomic research carried out with Lactobacillus delbrueckii subsp. bulgaricus showed that several proteins were strongly expressed in the lag phase, although only one protein (phosphoryl carrier protein HPr) was positively identified (30). Expression studies of Saccharomyces cerevisiae revealed about 20 proteins induced in the lag phase, which were primarily involved in protein synthesis, biosynthesis of amino acids, and carbohydrate metabolism (2, 3). A recent transcriptional study of Bacillus licheniformis based on 250 DNA probes exposed at least 15 differentially expressed genes of diverse functions in the lag phase (14).In this work, we investigated protein and gene expression in Lactococcus lactis subsp. lactis CNRZ 157 in the lag phase in comparison to the exponential and stationary growt...

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Section: Discussionmentioning

confidence: 81%

Differential Expression of Proteins and Genes in the Lag Phase of Lactococcus lactis subsp. lactis Grown in Synthetic Medium and Reconstituted Skim Milk

Larsen

Boye

Siegumfeldt

et al. 2006

Appl Environ Microbiol

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“…Formylation of initiator tRNA f Met was performed in parallel by adding N 10 -formyltetrahydrofolic acid (0.3 mM final) and purified histidine-tagged E. coli methionyl tRNA formyltransferase to the tRNA f Met aminoacylation reaction (Ramesh et al 1997). Aminoacylation reactions were stopped by the addition of sodium acetate (pH 5.2) to 300 mM and placed on ice.…”

Section: Aminoacylation Of Trnamentioning

confidence: 99%

Ribose 2′-hydroxyl groups in the 5′ strand of the acceptor arm of P-site tRNA are not essential for EF-G catalyzed translocation

Feinberg

Joseph²

2006

RNA

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The coupled movement of tRNA-mRNA complex through the ribosome is a fundamental step during the protein elongation process. We demonstrate that the ribosome will translocate a P-site-bound tRNA Met with a break in the phosphodiester backbone between positions 17 and 18 in the D-loop. Crystallographic data showed that the acceptor arms of P-and E-site tRNA interact extensively with the ribosomal large subunit. Therefore, we used this fragmented P-site-bound tRNA Met to investigate the contributions of single 2¢ -hydroxyl groups in the 5¢ strand of the acceptor arm for translocation into the ribosomal E-site. EF-G-dependent translocation of the tRNAs was monitored using a toeprinting assay and a fluorescence-based rapid kinetic method. Surprisingly, our results show that none of the 2¢ -hydroxyl groups in the 5¢ strand of the acceptor arm of P-site-bound tRNA Met between positions 1-17 play a critical role during translocation. This suggests that either these 2¢ -hydroxyl groups are not important for translocation or they are redundant and the three-dimensional shape of the P-site tRNA is more important for translocation.

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“…We investigated the in vivo state of the cytoplasmic yeast initiator tRNA in cells expressing the six-His-tagged wild-type MTF and the R42L mutant MTF. Previous studies had shown that introduction of the polyhistidine tag does not affect the activity of MTF (44). The R42L mutant MTF is ϳ1,400-fold less active in formylation of the E. coli initiator tRNA (45,46) and is used as a negative control here.…”

Section: Resultsmentioning

confidence: 99%

“…The E. coli MTF gene was cloned in the yeast expression vector YEp352-GAL1 (37). The wild-type and mutant MTF genes were amplified by PCR with the plasmids pQE16FMTp and pQE16FMTpR42L (44,45) as templates and the oligonucleotides 5Ј-ATCGGATCCAAAAAAAA TGTCAGAATCACTACGTATTATT-3Ј containing a BamHI site as the forward primer and 5Ј-CTCAGCTAATTAAGCTTAGTG-3Ј containing a HindIII site as the reverse primer. The PCR products coding for MTF-six-His fusion proteins were digested with BamHI and HindIII and cloned into the respective sites of YEp352-GAL1 to obtain YEp.MTF WT (wild type) and YEp.MTF R42L.…”

mentioning

confidence: 99%

Expression of Escherichia coli Methionyl-tRNA Formyltransferase in Saccharomyces cerevisiae Leads to Formylation of the Cytoplasmic Initiator tRNA and Possibly to Initiation of Protein Synthesis with Formylmethionine

Köhrer

RajBhandary

2002

Molecular and Cellular Biology

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Protein synthesis in eukaryotic cytoplasm and in archaebacteria is initiated with methionine, whereas, that in eubacteria and in eukaryotic organelles, such as mitochondria and chloroplasts, is initiated with formylmethionine. In view of this clear distinction, we have investigated whether protein synthesis in the eukaryotic cytoplasm can be initiated with formylmethionine, and, if so, what the consequences are to the cell. For this purpose, we have expressed in an inducible manner the Escherichia coli methionyl-tRNA formyltransferase (MTF) in the cytoplasm of the yeast Saccharomyces cerevisiae. Expression of active MTF, but not of an inactive mutant, leads to formylation of methionine attached to the yeast cytoplasmic initiator tRNA to the extent of about 70%. As a consequence, the yeast strain grows slowly. Coexpression of the E. coli polypeptide deformylase (DEF), which removes the formyl group from the N-terminal formylmethionine in a polypeptide, rescues the slow-growth phenotype, whereas, coexpression of an inactive mutant of DEF does not. These results suggest that the cytoplasmic protein-synthesizing system of yeast, like that of eubacteria, can at least to some extent utilize formylated initiator Met-tRNA to initiate protein synthesis and that initiation of proteins with formylmethionine leads to the slow-growth phenotype. Removal of the formyl group in these proteins by DEF would explain the rescue of the slow-growth phenotype.Protein synthesis in eubacteria, mitochondria, and chloroplasts is initiated with formylmethionine (20,21,41). Of the two classes of methionine tRNAs present in eubacteria, mitochondria, and chloroplasts, tRNA Met and tRNA f Met , the initiator methionyl-tRNA (Met-tRNA f Met ) is specifically formylated by the enzyme methionyl-tRNA formyltransferase (MTF) to generate formylmethionyl-tRNA (fMet-tRNA f Met ). The formyl group acts as a positive determinant for selection of the fMet-tRNA f Met by the initiation factor IF2 (54) and as a second negative determinant for blocking the binding of the tRNA to the elongation factor. Formylation of the initiator Met-tRNA f Met is important for protein synthesis in Escherichia coli, mutant initiator tRNAs defective in formylation are extremely poor in initiation of protein synthesis, and a strain of E. coli carrying disruptions in the MTF gene has severe growth defects (12,33,59).In contrast to the situation with eubacteria, mitochondria, and chloroplasts presented above, in the cytoplasm of eukaryotes and in archaebacteria, there is no formylation of the initiator tRNA (tRNA i Met ), and the Met-tRNA i Met is utilized directly to initiate protein synthesis with methionine (42, 47). However, the cytoplasmic initiator Met-tRNA i Met of yeast contains some of the determinants in the tRNA (Fig. 1) important for recognition by E. coli MTF (12a, 23) and can therefore be formylated in vitro by E. coli MTF (43). The resulting fMet-tRNA i Met initiates protein synthesis in cell extracts of both E. coli and rabbit reticulocytes, suggesting that the eu...

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Suppressor mutations in Escherichia coli methionyl-tRNA formyltransferase: Role of a 16-amino acid insertion module in initiator tRNA recognition

Cited by 29 publications

References 44 publications

Differential Expression of Proteins and Genes in the Lag Phase of Lactococcus lactis subsp. lactis Grown in Synthetic Medium and Reconstituted Skim Milk

Differential Expression of Proteins and Genes in the Lag Phase of Lactococcus lactis subsp. lactis Grown in Synthetic Medium and Reconstituted Skim Milk

Ribose 2′-hydroxyl groups in the 5′ strand of the acceptor arm of P-site tRNA are not essential for EF-G catalyzed translocation

Expression of Escherichia coli Methionyl-tRNA Formyltransferase in Saccharomyces cerevisiae Leads to Formylation of the Cytoplasmic Initiator tRNA and Possibly to Initiation of Protein Synthesis with Formylmethionine

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